1. Field of the Invention
The process of high-throughput screening (HTS) is central to the objectives of the pharmaceutical industry, i.e. to discover, develop and market new drugs (Lutz et al, (1996) Drug Discovery Today, 1(7), 277-86). In the HTS process, drug candidates are screened for possible effects in biological systems. Increasingly, there is a drive to test larger numbers of compounds in each screen, and screening assays examining 100,000 compounds or more are typical. This requires highly sophisticated robotic automation and instrumentation to achieve efficient levels of throughput. In general, modem screening techniques utilise multiwell plate technologies to allow transfer of the many thousands of assays between the various stages in the procedure. Such plates may contain between 96 and 1536 or more individual wells, where each well contains the same reagents as all other wells in the screen, except for the individual compounds under test which are each present in only one well. The standard format and layout of the multiwell plates allows fast robotic handling and liquid dispensing devices to be used to maximise throughput.
2. Description of Related Art
In many HTS applications the rate limiting step occurs in assay analysis at the stage of detecting and measuring the signal from the label used in the assay. This step is a serial process, each well of the multiwell plate being measured in turn. Such measurements typically require from one to several seconds to perform, with the consequence that the time taken to analyse a multiwell plate can be considerable.
Flow Cytometry (Parks, D. R. and Herzenberg, L. A. 1984, Methods in Enzymology 108, 197-241) is a technique for analysing cells or particles according to their size and fluorescence. The cells or particles are carried by a thin rapidly moving stream of liquid which is transected by light beam(s) from one or more lasers or other light sources. Photo-detectors register light-scattering and fluorescence arising from a cell or particle passing through a light beam and the resulting electronic signals are processed to yield analytical data. In contrast to the slow data acquisition time of multiwell plate readers, instrumentation for flow cytometry enables very rapid analysis of many thousands or millions of cells or other particles in a high speed stream of liquid and is typified by very fast measurement times, for example of the order of 1 μsec/event.
Flow cytometry has other characteristics which make it favourable for analysis in HTS. Firstly, the very small analysis volumes required are compatible with the current trend to scale down assays as a means of increasing throughput. Secondly, flow cytometry is inherently an homogeneous measurement system, i.e. measurement of the fraction of a specific fluorescent dye-labelled ligand in a particular state can be accomplished without the need to physically separate that type of fluorescent dye from the total type. In HTS applications, this is a desirable property as it removes the need for washing or separation stages to isolate the desired type of label prior to measurement. Flow cytometry has been extensively used for diagnostic assays to measure a wide range of analytes in blood and other biological fluids, for example in immunotyping and measurement of cell surface antigens associated with HIV infection (Patterson, B. K. et al J. Virology, (1995) 69(7) 4316-4322). Despite its inherent advantages however, flow cytometry is disadvantaged by low throughput rates which are a consequence of serial processing. While read times are very fast, allowing many thousands of events to be analysed/second within a single assay, there is a considerable delay between samples which currently limits overall throughput to <100 separate analyses/hour.
A desire to have a higher throughput in these applications has led to the development of multiplex methods which allow more than one analyte to be measured simultaneously by flow cytometry. Multiplexing is achieved by carrying out solid phase linked assays using plastic or latex beads as assay substrates. By using a number of discrete bead types which are individually distinguishable from each other, where each bead type carries reagents for one assay, standard flow cytometer instrumentation may be used both to identify the bead type and to measure the assay signal associated with each bead, and therefore to perform several tests in parallel on a single sample, for example to measure the presence of multiple analytes in human sera (McHugh T. M., 1994, Methods in Cell Biology 42, 575-595). Discrimination between bead types can be achieved by size (Frengen J. et a/, 1995, Journal of Immunological Methods, Volume 178, p141). by colour or fluorescence (Fulwyler M. J. UK Patent 1,561,042) or by electronic means (Mandecki W. U.S. Pat. No. 5,641,634).
Multiplexing of flow cytometry assays introduces an element of parallel processing into an otherwise serial process, so that while the delay between samples remains as before, the amount of information gathered from each sample is increased several fold giving a resulting increase in data acquisition rates. This is ideal for measurement of multiple analytes in a single sample, i.e. ‘one sample, many tests’. However, the requirements of high throughput screening, i.e. ‘one test, many samples’, are the reverse. In HTS assays it is a requirement that there must always be separation of assays to allow the effects of individual compounds within the screen to be determined. Consequently, methods previously described for multiplex diagnostic analyses by flow cytometry are not applicable to HTS assays.